DTIC ADA628281: Susceptibility of Oral Bacteria to an Antimicrobial Decapeptide

12 Pages·0.24 MB·English

by  Defense Technical Information Center

JournalofMedicalMicrobiology(2003),52,1083–1093 DOI10.1099/jmm.0.05286-0
Susceptibility of oral bacteria to an antimicrobial
decapeptide
S.P.Concannon,1†T.D.Crowe,1 J.J.Abercrombie,1 C.M.Molina,1 P.Hou,1
D.K. Sukumaran,2 P. A. Raj3 and K.-P. Leung1
Correspondence 1MicrobiologyBranch,USArmyDentalResearchDetachment,WalterReedArmyInstituteof
K.-P.Leung Research,GreatLakes,IL60088,USA
[email protected] 2DepartmentofChemistry,StateUniversityofNewYorkatBuffalo,Buffalo,NY14260,USA
3SchoolofDentistry,MarquetteUniversity,Milwaukee,WI53233,USA
Naturallyoccurringantimicrobialpeptideshaveemergedasalternativeclassesofantimicrobials.In
general,theseantimicrobialpeptidesexhibitselectivityforprokaryotesandminimizetheproblemsof
engenderingmicrobialresistance.Asanalternativemethodtosearchformoreeffectivebroad-
spectrumpeptideantimicrobials,investigatorshavedevelopedpeptidelibrariesbyusingsynthetic
combinatorialtechnology.Anoveldecapeptide,KKVVFKVKFK(KSL),hasbeenidentifiedthat
showsabroadrangeofantibacterialactivity.Thepurposeofthisstudywastotesttheefficacyofthis
antimicrobialpeptideinkillingselectedstrainsoforalpathogensandresidentsalivabacteria
collectedfromhumansubjects.CytotoxicactivityofKSLagainstmammaliancellsandthestructural
featuresofthisdecapeptidewerealsoinvestigated,thelatterbyusingtwo-dimensionalNMRin
aqueousandDMSOsolutions.MICsofKSLforthemajorityoforalbacteriatestedinvitroranged
from3to100(cid:1)gml(cid:1)1.MinimalbactericidalconcentrationsofKSLwere,ingeneral,withinonetotwo
dilutionsoftheMICs.KSLexhibitedanED (thedoseatwhich99%killingwasobservedafter
99
15minat378C)of6.25(cid:1)gml(cid:1)1againstselectedstrainsofLactobacillussalivarius,Streptococcus
mutans,StreptococcusgordoniiandActinobacillusactinomycetemcomitans.Inaddition,KSL
damagedbacterialcellmembranesandcaused1.05logunitsreductionofviabilitycountsofsaliva
bacteria.InvitrotoxicitystudiesshowedthatKSL,atconcentrationsupto1mgml(cid:1)1,didnotinduce
celldeathorcompromisethemembraneintegrityofhumangingivalfibroblasts.NMRstudies
suggestthatKSLadoptsanÆ-helicalstructureinDMSOsolution,whichmimicsthepolaraprotic
membraneenvironment,whereasitremainsunstructuredinaqueousmedium.Thisstudyshowsthat
Received 14April2003 KSLmaybeausefulantimicrobialagentforinhibitingthegrowthoforalbacteriathatareassociated
Accepted 24August2003 withcariesdevelopmentandearlyplaqueformation.
INTRODUCTION classes of peptide antibiotics that exhibit selectivity for
prokaryotes and minimize the problems of introducing
We have witnessed the declining efficacy of conventional
microbial resistance (Boman, 1998; Hancock & Lehrer,
antibioticsinrecentyears,duetotheprogressiveincreaseand
1998;Hancock&Chapple,1999;Nizetetal.,2001;Zasloff,
proliferationofantibiotic-resistantorganisms(Davies,1994;
2002).Thesepeptideantibioticsinteractdirectlywithmicro-
Schutze et al., 1994). The discovery of a large number of
bial surfaces, often leading to the formation of pores or in
naturally occurring invertebrate and vertebrate antimicro-
some way compromising membrane permeability (Zasloff,
bial peptides has resulted in the emergence of alternative
1992;Hancock,1997a;Hancock&Rozek,2002;Koczulla&
Bals, 2003; Yeaman & Yount, 2003). Aside from their
†Presentaddress:WyethBioPharma,StLouis,MO63134,USA. antimicrobialactivities,someofthesepeptidesalsopossess
Abbreviations:CD,circulardichroism;DMF,N,N-dimethylforamide;DQF- otherbiologicalactivitiesthathaveimpactsoncellprolifera-
COSY,doublequantum-filteredcorrelatedspectroscopy;Fmoc,9- tion, immune induction, cytokine release, chemotaxis and
fluorenylmethoxycarbonyl;HGF,humangingivalfibroblasts;LDH,lactate
tissuerepair(Batemanetal.,1991;Elsbach,2003;Koczulla&
dehydrogenase;MALDI-TOF,matrix-assistedlaserdesorption/
Bals,2003;Koczullaetal.,2003).
ionization–timeofflight;MBC,minimalbactericidalconcentration;MTT,3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;NOE,nuclear
Ingeneral,antimicrobialpeptidesexhibitdiversestructures;
Overhausereffect;NOESY,nuclearOverhausereffectspectroscopy;
RMSD,rootmeansquaredeviation;TOCSY,totalcorrelatedspectroscopy. however,mostarecationicamphiphilicmoleculesbecauseof
05286 PrintedinGreatBritain 1083
Report Documentation Page Form Approved
OMB No. 0704-0188
Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and
maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,
including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington
VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it

does not display a currently valid OMB control number.
1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
01 DEC 2003 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Susceptibility of oral bacteria to an antimicrobial decapeptide
5b. GRANT NUMBER
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Concannon S. P., Crowe T. D., Abercrombie J. J., Molina C. M., Hou P.,
5e. TASK NUMBER
Sukumaran D. K., Raj P. A., Leung K. P.,
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United States Army Institute of Surgical Research, JBSA Fort Sam REPORT NUMBER
Houston, TX 78009
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON

a. REPORT b. ABSTRACT c. THIS PAGE SAR 11
unclassified unclassified unclassified
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
S.P.Concannonandothers
the presence of arginine and lysine residues and can be cillus salivarius ATCC 29602, Lactobacillus acidophilus ATCC 43571
grouped into four or five different structural categories. and Actinobacillus actinomycetemcomitans ATCC 43718. S. mutans
These include: (a) cysteine-rich, amphiphilic (cid:1)-sheet pep- strainLT11waskindlyprovidedbyLinTao,UniversityofIllinoisat
tides(Æ-and(cid:1)-defensins,protegrinsandtachyplesins);(b) Chicago,IL,USA.S.gordoniiDL1andS.sanguinisSK36wereprovided
byJohnCisar,NationalInstituteofDentalandCraniofacialResearch,
cysteine–disulfideringpeptideswithorwithoutamphiphilic
NIH,Bethesda,MD,USA.
tails (bactenecin, ranalexin and brevinins); (c) amphiphilic
Æ-helical peptides without cysteine (magainins and cecro-
pins); and (d) linear peptides (Bac 5, Bac 7, PR39 and Synthesis of the antimicrobial decapaptide KSL. KSL
indolicidin) with one or two predominant amino acids (KKVVFKVKFK-NH )wassynthesizedbystandardsolid-phasepro-
2
(proline or tryptophan) (Hancock et al., 1995; Hancock, ceduresasdescribedbyHongetal.(1998)byusing9-fluorenylmethox-
1997b;Hancock&Lehrer,1998;Hendersonetal.,1998). ycarbonyl (Fmoc) chemistry on a model 90 automatic peptide
synthesizer (Advanced ChemTech). The peptide was synthesized on
Manysyntheticanaloguesofthesepeptideshavebeencreated Rinkamidemethylbenzhydrylamine(MBHA)resin(AnaSpec)withthe
inattemptstoimprovetheantimicrobialactivityofsomeof firstN-Fmoc-protectedLysattached.Sequentialcouplingofprotected
Fmoc amino acids, which included Phe, Lys and Val, was done in
thesenaturallyoccurringantibacterialpeptides(Wadeetal.,
N9-tetramethyluronium tetrafluoroborate (TBTU) dissolved in N,N-
1992;Tamamuraetal.,1995;Helmerhorstetal.,1997;Fuchs
dimethylforamide (DMF) that contained 0.55M N,N9-diisopropyl-
etal.,1998;Chenetal.,2000;Moscaetal.,2000;Rothsteinet
ethylamine(DIEA)(allfromAdvancedChemTech).Piperdine[20%
al.,2001).Forexample,Dhvar5,ananalogueofhistatin5, (v/v)inDMF]wasusedtoremovetheN-terminalFmocmoietyfrom
one of the antimicrobial histatin peptides that are derived the growing peptide prior to subsequent coupling. Completion of
from saliva (Helmerhorst et al., 1999; Mickels et al., 2001) couplingreactionswasassessedbytheninhydrintestofKaiseretal.
and IB-367, an analogue of protegrins, antimicrobial pep- (1970).Cleavageofthepeptidefromtheresinandthedeprotectionof
sidechainsweredonebyusingamixtureof95%trifluoroaceticacid
tidesthatwereisolatedfromporcineleukocytes(Zhaoetal.,
and 5% ethane dithiol. Synthetic peptides were purified by reverse-
1994;Chenetal.,2000;Moscaetal.,2000),aremoreeffective
phase HPLC (series 1100; Hewlett Packard) by using a Vydac C18
ininhibitingbacterialgrowthandaresynthesizedmoreeasily
column. Peptide purity was confirmed by MALDI-TOF (matrix-
thantheirnativecounterparts. assistedlaserdesorption/ionization–timeofflight)MS,asperformed
by the laboratories of AnaSpec. The final product was stored in
Inadditiontotheidentificationofthesenaturalantimicro- lyophilizedformat(cid:1)208Cuntiluse.
bial peptides and their synthetic analogues, investigators
have developed peptide libraries by using synthetic combi-
natorial technology (Blondelle et al., 1994, 1996a, b; NMRstructuralanalysis.Purifiedpeptide(7mg)wasdissolved in
Blondelle & Houghten, 1996; Hong et al., 1998; Boggiano 630(cid:1)l double-distilled water and 70(cid:1)l 2H2O (Cambridge Isotope
et al., 2003) to search for more effective broad-spectrum Laboratories) at a peptide concentration of (cid:2)5 mM. The pH of the
aqueous peptide solution was3.8.For NMR experiments in DMSO,
peptide antimicrobials. Using this technology, Hong et al.
peptide(7mg)wasdissolvedin700(cid:1)l99.9%(C2H ) SO(Cambridge
(1998)identifiedanoveldecapeptide,KKVVFKVKFK,that 3 2
Isotope Laboratories). One-dimensional (1D) and two-dimensional
shows a broad range of antibacterial activity. This peptide
(2D) NMR experiments used for conformational analyses were per-
antimicrobialiseffectiveininhibitingthegrowthofmethi- formedat308Cinthesesolvents.AllNMRexperimentswerecarriedout
cillin-resistantStaphylococcusaureus(MRSA),Pseudomonas at500MHzonaVarianUnityInovaspectrometerequippedwithaSUN
aeruginosaandanumberofenterics.Inaddition,thispeptide Sparcstation20.1D-NMRspectrawererecordedwithaspectralwidthof
inhibitsthegrowthofCandidaalbicansirreversibly,suggest- 5000Hzandarelaxationdelaytimeof2.5s,using8Kdatapointszero-
filledto32KbeforeFouriertransformation.All2Dexperimentswere
ing that this agent also possesses antifungal activity (Hong
multipliedbyaphase-shiftedsinebellfunctioninbothdimensionsand
etal.,1998).
zero-filledpriortoFouriertransformation,inordertoachieveappro-
priateresolutionineachdimension.NuclearOverhausereffectspectro-
Inthisreport,wedescribethethree-dimensionalstructureof
scopy (NOESY), double quantum-filtered correlated spectroscopy
this decapeptide in aqueous solution and in membrane
(DQF-COSY)andtotalcorrelatedspectroscopy(TOCSY)experiments
environments, the effects of this antimicrobial peptide on were performed by using standard methods, as described in our
oral bacteria that are involved in the formation of dental previous studies (Raj et al., 1998, 2000a). Coupling constant
plaqueandthedevelopmentofcariesandtheinteractionsof (JNH–CÆH)valuesweredeterminedeitherfromtheresolved1Dspectra
thismoleculewithhumangingivalfibroblasts(HGF)forany (digital resolution, 0.1 Hz) or from high-resolution DQF-COSY
spectra. Hydrogen–deuterium (1H–2H) exchange of amide groups
cytotoxiceffectsassociatedwiththispeptide.
and variable temperature experiments were performed as described
previously(Rajetal.,1998).
METHODS Forstructurecalculation,1H–1Hdistancesforstructuredetermination
were deduced from nuclear Overhauser effect (NOE) cross-peak
Bacterialstrains.StrainsusedinthisstudyincludedAmericanType intensitiesinthe2D-NOESYspectrum,obtainedwith150msmixing
Culture Collection (ATCC) and laboratory strains of different oral time in water. The C(cid:1)H/C(cid:1)H9 cross-peak of Phe5 was selected as the
bacteria. These included: Actinomyces naeslundii strain T14V-J1, referencetocalibratetheintensitiesagainstknowndistances.Dihedral(cid:2)
Actinomyces israelii ATCC 10049, Streptococcus mutans strains LT11 anglerestraintswereobtainedfromJNH–CÆH viatheKarplusequation
andATCC25175T,StreptococcussobrinusATCC33478T,Streptococcus (Pardietal.,1984).Atotalof135NOEconstraints,dÆN (i,i+1),dÆN
gordoniistrainsDL1andATCC51656,Streptococcussanguinisstrains (i, i), d(cid:1)N (i,i), dÆ(cid:3) (i, i+3), dÆÆ (i, i+1)and side chain interproton
SK36andATCC10556T,StreptococcussalivariusATCC9222,Strepto- distanceswereusedasparametersforstructuredetermination.Distance
coccusmitisATCC15913,StreptococcusoralisATCC35037T,Lactoba- geometry calculations were performed on a Silicon Graphics 4D/35
1084 JournalofMedicalMicrobiology52
Bactericidaldecapeptide
workstation. Restrained energy minimization and structure analysis mycetemcomitans ATCC 43718 and L. salivarius ATCC 29602, were
werecarriedoutbyusingtheSYBYL6.02molecularmodellingpackage testedfortheirsusceptibilitytoKSLbythebactericidalassay.
(TriposAssociates)onanEvans&SutherlandESV3workstation.
Media,cultureconditionsandinvitrosusceptibilitytests.Todd– Invitrokillingofsalivabacteria.Thekillingassayofsalivabacteria
Hewittbroth(THB),brainheartinfusion(BHI)broth,Todd–Hewitt was done according to procedures established by Helmerhorst et al.
agar(THA),trypticasesoyagar(TSA),lactobacilliMRS(MRS)broth, (1999) with a slight modification. Unstimulated saliva was collected
ActinomycesbrothandMueller–Hintonbroth(MHB)werepurchased fromfourhealthyindividualswhohadrefrainedfromeatingforatleast
from Becton Dickinson. Blood agar plates (BAP) were prepared by 2h.ThestudywasapprovedbytheInstitutionalReviewBoardofthe
supplementingTSAwith5%sheepblood(PMLMicrobiologicals).All Walter Reed Army Institute of Research and informed consent and
cultures,exceptforActinobacillusactinomycetemcomitans,S.mitisand sample donation consent were obtained from all volunteers. Pooled
Actinomyces israelii, were grown at 378C in room air. Actinobacillus salivawasinitiallyspunat500r.p.m.inanEppendorfcentrifuge(model
actinomycetemcomitans cultures were grown on BAP or in MHB 5810R)for10minat48Ctoremoveepithelialcellsandmucus.Saliva
supplementedwith15(cid:1)ghaeminml(cid:1)1(Sigma),15(cid:1)gNADml(cid:1)1(Sig- bacteriawerecollectedbyspinningthesupernatantat4000r.p.m.for
ma)and5%yeastextract(BectonDickinson)at378Cin5%CO2.S. 15minat48C.Thepelletwaswashedthreetimesin10mMpotassium
mitis was grown on BAP or in BHI broth at 378C in 5% CO2. phosphate buffer (PPB) and suspended in the same buffer to give
Actinomycesisraeliiwasgrownanaerobicallyinananaerobicchamber approximately1.03107 cellsml(cid:1)1.Bacterialsuspension(250(cid:1)l)was
(CoyLaboratoryProducts)ina5%CO2/10%H2/85%N2atmosphere. mixedwith250(cid:1)lpeptidetoobtainfinalpeptideconcentrationsof12.5,
MICsweredeterminedasdescribedbyFuchsetal.(1998)withaslight 25,50,100and200(cid:1)gml(cid:1)1.Afterincubationofthemixtureat378Cfor
modification.ProcedureswerebasedontheNationalCommitteefor 30min,cellswerespundowntoremoveKSL,washedonceinPBS(pH
ClinicalLaboratoryStandardsbrothmicrodilutionmethod.MHBwas 7.4)andsuspendedinPBSbeforespiral-plating(50(cid:1)l)ofthetreated
usedasthemainassaymediumformostofthetestedorganisms.Freshly and untreated (negative control; exposed to buffer only) cells in
grownculturesatexponentialphasewereusedastheinoculum.Bacteria differentdilutionsonBAP.Salivabacteriaexposedto0.12%aqueous
werecentrifugedat4000r.p.m.for15minat48C,suspendedin23 chlorhexidineservedaspositivecontrols.
concentrated medium and adjusted to 43106 c.f.u. ml(cid:1)1 in 23
concentratedmedium.Aqueouspeptidesolution(100(cid:1)l)wasadded
toeachwellofa96-well,flat-bottomedplate(BectonDickinson).The
peptidesolutionwasseriallydiluted(twofold)withsteriledistilledwater AssessingviabilityofKSL-treatedsalivabacteria.ALIVE/DEAD
in the wells, with final peptide concentrations ranging from 3.13 to BacLightBacterialViabilitykit(MolecularProbes)wasusedtoassess
200(cid:1)gml(cid:1)1. After dispensing 100(cid:1)laliquots ofbacterial suspension theviabilityandstatusofmembraneintegrityinsalivabacteriatreated
intothewells,the96-wellplateswereincubatedat378Cfor24–48hin withaqueousKSL(200(cid:1)gml(cid:1)1).BacLightassaysolutionwasprepared
room air, CO or anaerobically. The MIC was defined as the lowest asdescribedbythemanufacturer.SalivabacterialsuspensioninPPB(50
concentration2ofthepeptidethatpreventedvisibleturbidity,asmeas- (cid:1)l)wasmixedwithanequalvolumeofaqueousKSLtoobtainafinal
uredat600nmbyusinganELISAreader(TitertekMultiskanMCC/ peptideconcentrationof200(cid:1)gml(cid:1)1.Bacterialsuspensionmixedwith
340). Visible turbidity was determined by the OD readings oftested steriledH2Owasusedasthenegativecontrol.BacLightsolution(1.5(cid:1)l)
samples that were significantly greater than that of the medium, i.e. was added to the mixture after incubation at 378C for 30min. The
background. Minimum bactericidal concentrations (MBCs) were reactionmixturewasincubatedfurtheratroomtemperatureinthedark
determined by spiral-plating (Spiral Plater Autoplate 4000; Spiral for15min.SampleswereobservedbyusingafluorescenceAxioplan2
Biotech)50(cid:1)lfromeachclearwell(>MIC)ontoBAP.Afterincubation imaging system (Zeiss) equipped with longpass and dual-emission
for24–48h,theMBCwasdeterminedasthelowestconcentrationthat filters(Chroma)forsimultaneousviewingoflive(stainedbySYTO9)
did not permit visible growth on the surface of the agar. For the anddead(stainedbypropidiumiodide)bacteria.
susceptibility study, a small peptide at 200(cid:1)gml(cid:1)1, which had a
sequenceofLYPQPYQPQYQQYTF,andamoxicillinat5(cid:1)gml(cid:1)1were
usedasnegativeandpositivecontrols,respectively.Thiscontrolpeptide
was the C-terminal sequence (29–43) of salivary statherin, which Invitrotoxicitystudies.HGF,obtainedfromtheATCC(Manassas,
showednoantimicrobialpropertiesinourpreviousstudies(datanot VA,USA),wereusedasthetargetforinvitrotoxicitystudies.HGFwere
shown). culturedinRPMI1640medium(GibcoBRL)thatcontained5%fetal
bovineserumat378CinaCO incubatorpriortoexposuretovarious
2
concentrationsofKSL,whichincludedconcentrationsthatwereatleast
Bactericidalassay.Thebactericidalassaywasperformedaccordingto tenfold(upto1mgml(cid:1)1)abovetheeffectiveantimicrobialdosesused
procedures described by Miyasaki et al. (1997, 1998) with a slight inthebactericidalassay.Cellsthatwereexposedtomediumaloneserved
modification. Briefly, bacterial suspension in Hanks’ balanced salt ascontrols.Untreatedandaffectedcellswereexaminedforviability,as
solution (HBSS; Sigma), pH 7.0, or in 0.01% BHI (Actinobacillus determinedbytheirabilitytoreduce3-(4,5-dimethylthiazol-2-yl)-2,5-
actinomycetemcomitans), was adjusted spectrophotometrically at diphenyltetrazoliumbromide(MTT)(Sigma)(Mosmann,1983),and
660nm to approximately 1.03107 cells ml(cid:1)1. Bacterial suspension theirmembraneintegrity,asafunctionoftheamountofcytoplasmic
(90(cid:1)l)wasmixedwith10(cid:1)lKSLatdifferentconcentrationsandthe lactate dehydrogenase (LDH) released into the medium (Decker &
reactions were incubated at 378C for 15min. The reaction was Lohmann-Matthes,1988).DetectionofcellularconversionofMTTto
terminatedbyadding900(cid:1)lice-coldHBSStothemixtureand50(cid:1)l water-insolublecolouredformazananddeterminationoftotal(cyto-
of each sample was spiral-plated on agar media, as specified in the plasmic and extracellular) and extracellular LDH of affected and
Results section. Susceptibility was determined by examining the log untreated cells were done according to the instructions of the
reduction in viability counts of organisms that had been exposed to manufacturer of a commercially available in vitro toxicity assay kit
differentconcentrationsofKSL.BacteriasuspendedinHBSSservedasa (Sigma).ReactionproductsoftheLDHassayweremeasuredspectro-
control. Bactericidal activity was also expressed as the 99% effective photometricallybyusingatestwavelengthof490nmandareference
dose(ED ),whichistheconcentrationoftheantimicrobialdecapep- wavelength of 690nm. For measuring acid/isopropanol-solubilized
99
tideatwhichthereisa2-logormorereductioninc.f.u.Fourstrains,S. formazan,atestwavelengthof570nmandareferencewavelengthof
mutansATCC25175T,S.gordoniiATCC51656,Actinobacillusactino- 630nmwereused.
http://jmm.sgmjournals.org 1085
S.P.Concannonandothers
RESULTS
Synthesisofpeptide
KSL, synthesized by using standard Fmoc chemistry, was
purified by reverse-phase HPLC and subjected to MALDI-
TOF MS to verify the integrity of the peptide. The mass
spectrum of KSL had an intense molecular ion at m/z
1249.80, which was consistent with its relative molecular
massof1250(datanotshown).
NMRstudiesofKSL
SequentialresonanceassignmentsofKSL. Assignments
of1Hresonanceswereaccomplishedbycombinedanalysesof
2D TOCSY and 2D NOE spectra. Identification of most of
the spin systems was achieved unambiguously from amide
proton-relayed TOCSY connectivities. Assignment of reso-
nances to individual amino acids was accomplished by
combined analyses of the NH–CÆH connectivities in the
fingerprint regions in the TOCSY and 2D NOE spectra, as
describedpreviously(Rajetal.,1996,1998,2000a).
Conformationalanalysisinaqueoussolution.Asummary
Fig.1.Summary of sequential and medium-range NOE data com-
ofobservedNOEconnectivities,temperaturecoefficientsof
piledfromtheNOESYspectraofKSLrecordedat308C:(a)inH O/
2
NHchemicalshiftsandcouplingconstant(JNH–CÆH)values 2H O,usingamixingtimeof150ms;and(b)in(C2H ) SO,usinga
2 3 2
areprovidedinFig.1.Temperaturecoefficientsofallamide
mixingtimeof200ms.ThicknessofbarsindicatesthattheNOEis
resonances provided in Fig. 1(a) are high (>0.0042 p.p.m. strong,mediumandweak,respectively. d,Slow-exchangingamide
K(cid:1)1) and the fast 1H/2H exchange rate observed for all NHs.
backboneamideresonances(Fig.1a)in65%2H Oprovides
2
evidence that the amide groups are not involved in any
intramolecularhydrogenbonding.PrevalenceofstrongÆN
(i,i+1)andweakÆN(i,i)NOEs(Fig.1a)andacontinuous HzforresiduesfromPhe5toLys10(Fig.1b),indicatingthe
stretchofweakandmedium(cid:1)N(i,i)andÆ(cid:1)(i,i)NOEs(Fig. presenceofasignificantpopulationofhelicalconformation
1a) in the absence of any observable NN NOE interactions inDMSOsolution.Thesummaryofsequentialandmedium-
indicatethatthebackbonedihedralanglesarepredominantly rangeNOEsobservedforKSL(Fig.1b)in(C2H ) SOshows
intheunfoldedextendedregionofthe(cid:2),łspace(Wu¨thrich, sequentialNN(i,i+1)connectivitiesandmediu3m2-rangeÆ(cid:1)
1986).TheJNH–CÆHvaluesprovidedinFig.1aare>7.3Hzfor (i, i+3) interactions that are characteristic of Æ-helical
allresiduesexceptLys2andPhe9.Couplingconstantsof7.3– conformations (Wu¨thrich, 1986; Dyson & Wright, 1991).
8.1HzwereobservedformostresiduesofKSL;thissuggests In addition, a complete set of weak ÆN (i, i+3) NOEs,
the existence of populations of unfolded, non-hydrogen- expectedforÆ-helicalconformation,wasobserved(Fig.1b).
bondedconformationsofcomparableenergywith(cid:2)values These NOE NN (i, i+1) values provide support for the
thatexceedthoseoftheregularhelicalregion.TheNMRdata prevalenceofathresholdpopulationofÆ-helicalconforma-
obtained provide evidence that KSL molecules remain tions.Collectively,theNMRdatasuggeststronglythatKSL
unstructuredinaqueoussolution. exists as Æ-helical conformers that involve Lys2–Lys10,
whicharestabilizedbysix(5!1)intramolecular hydrogen
ConformationalanalysisinDMSOsolution.In(C2H ) SO, bondsin(C2H ) SO.
3 2 3 2
loweredtemperaturecoefficientsofbackboneamidegroups
(<0.0029p.p.m.K(cid:1)1),exceptforthefirstfourresidues,have MolecularstructureofKSLinDMSOsolution.Theaverage
beenobserved(Fig.1b).Theseresultssuggestthatsixamide structureofafamilyof16conformersobtainedafterenergy
NH groups from Phe5 to Lys10 could be involved in minimizationofthedistancegeometryalgorithmforNMR
intramolecular hydrogen bonding, whereas the amide NH applications(DIANA)structures,asdescribedintheMeth-
groupsofLys2,Val3andVal4arewellexposedtothesolvent. odssection,isshowninFig.2(left).AviewoftheÆ-helical
Slow 1H/2H exchange of the Phe5–Lys10 NH groups (Fig. structurealongthehelixaxisisalsoprovidedinFig.2(right).
1b)alsosuggeststhattheamideNHsofPhe5–Lys10maybe The calculated mean pairwise root mean square deviation
inaccessible to the solvent and are probably involved in (RMSD)andtheassociatedSDsare1.78(0.32)A˚ forallatoms
intramolecular hydrogen bonds (Wu¨thrich, 1986). In and 1.12 (0.18) A˚ for the backbone (without taking into
(C2H3)2SO, the JNH–CÆH values were in the range 5.2–5.9 considerationtheN-terminalresidue,Lys1).Whentakingall
1086 JournalofMedicalMicrobiology52
Bactericidaldecapeptide
Fig.2.A perspective view of the averaged Æ-
helicalstructureofKSLwiththeN-terminusat
thetopandtheC-terminusatthebottom(left).A
viewoftheÆ-helicalstructureofKSLalongthe
helix axis, illustrating the weak amphipathic
natureofthemolecule(right).Forclarity,hydro-
genatomsarenotincluded.
residuesintoaccount,RMSDsare2.38(0.47)and1.57(0.26) thefacultativelyanaerobicoralorganismstested.Bycontrast,
A˚ for all atoms and backbone atoms, respectively. The thecontrolpeptideat200(cid:1)gml(cid:1)1didnotinhibitthegrowth
structure of KSL is a single-stranded, Æ-helix stabilized by of any organisms tested. Bacteria grown in medium that
six intramolecular (5!1) hydrogen bonds formed by the contained the control peptide grew to the same extent as
backboneamideNHgroupsofPhe5–Lys10. organisms grown in medium alone (data not shown). KSL
was effective in inhibiting growth of most of the primary
colonizers involved in the initiation of plaque formation,
Invitrosusceptibilityoforalbacteria
which included Actinomyces naeslundii, S. gordonii and S.
MICs and MBCs of KSL for the majority of oral bacteria sanguinis,asdemonstratedbythebrothmicrodilutionassay.
tested in MHB or other modified broth were determined GrowthofthecariogenicbacteriaS.mutansATCC25175T,S.
(Table1).MICsrangedfrom3.13to100(cid:1)gml(cid:1)1formostof sobrinus and L. acidophilus was also inhibited effectively by
Table1.InvitrosusceptibilityoforalbacteriatoKSL
Bacterialstrain* MIC((cid:1)gml21)† MBC((cid:1)gml21)
ActinomycesnaeslundiiT14V-J1 3.13 12.5
ActinomycesisraeliiATCC10049 6.25 12.5
S.mutans:
ATCC25175T 6.25 12.5
LT11 25 100
S.gordonii:
ATCC51656 50 100
DL1 50 100
S.sanguinis:
ATCC10556T 25 100
SK36 25 50
S.oralisATCC35037T 100 200
S.sobrinusATCC33478T 25 50
S.salivariusATCC9222 50 100
S.mitisATCC15913 25 50
ActinobacillusactinomycetemcomitansATCC43718 100 200
L.salivariusATCC29602 3.13 12.5
L.acidophilusATCC43571 3.13 12.5
*Thenumberofcellsusedintheseassayswas43106ml(cid:1)1,withtheexceptionofActinobacillusandS.
mutansLT11,forwhich43107cellsml(cid:1)1wereused.
†MHBwasusedforthedeterminationofmostoftheMICs.ForS.mitisandActinobacillus,BHIand50%
BHIwereusedastheassaymedium,respectively.10%MRSand20%Actinomycesbrothwereusedasthe
assaymediumforLactobacillusstrainsandActinomycesisraelii,respectively.Cultureconditionswerethe
sameasthosedescribedinMethods.
http://jmm.sgmjournals.org 1087
S.P.Concannonandothers
KSLatconcentrationsof,25(cid:1)gml(cid:1)1.Ontheotherhand, Treatmentofthesalivabacteriawithafinalconcentrationof
KSL possessed less growth-inhibitory activity against Acti- 200(cid:1)gKSLml(cid:1)1 resultedina1.05-logreductioninfaculta-
nobacillus actinomycetemcomitans and S. oralis. In general, tive anaerobes that were present in thesaliva, comparedto
many of the MBCs of KSL for the organisms tested were PBS-treatedsalivasamples(negativecontrol).Asapositive
withinonetotwodilutionsoftheMICs. control,chlorhexidine(0.12%)causeda.3-logreductionin
facultativelyanaerobicsalivaorganisms(datanotshown).
Bactericidalassay(reductionofviablecounts)
InadditiontothedeterminationsofMICsandMBCsofKSL, Fluorescence microscopy of samples stained with LIVE/
log reductions in viable counts of selected oral bacteria DEAD BacLight assay solution showed that many of the
causedbyKSLwerealsodetermined.Thesewerecompared saliva bacteria in the control sample (bacteria treated with
to c.f.u. of organisms incubated in buffer, which served as dH O) fluoresced green (Fig. 5a). On the other hand,
2
controls (shown as 0(cid:1)gKSLml(cid:1)1 in Fig. 3). With the bacterialsuspensionthathadbeentreatedwithKSLshowed
exception of Actinobacillus actinomycetemcomitans, for a significant number of saliva bacteria that fluoresced red
whichtheassayswereperformedin0.01%BHI,bactericidal (Fig.5b)
action of KSL was determined in isotonic HBSS after
incubation with the targeted bacteria for 1h at 378C. KSL
at 6.25(cid:1)gml(cid:1)1, which was determined to be the 99%
effectivedose(ED ),causedmorethan2logreductionsin ViabilityofKSL-treatedHGF
99
viable counts of the organisms tested, which included L.
KSL at concentrations up to 1mgml(cid:1)1 did not inducecell
salivariusATCC29602,S.mutansATCC25175T,S.gordonii
deathofKSL-treatedfibroblasts,asindicatedbytheabilityof
ATCC 51656 and Actinobacillus actinomycetemcomitans
treatedcellstoreduceMTTatlevelsthatweresimilartothose
ATCC43718(Fig.3).
ofuntreatedHGF(Fig.6a).Also,KSLdidnotcompromise
the membrane integrity of KSL-treated fibroblasts. Similar
Viabilityandmembraneintegrityofsalivabacteria
levels of extracellular LDH were observed among KSL-
treatedwithKSL
treatedcellsanduntreatedHGF(Fig.6b).HGFcontaineda
There was a significant reduction in viable counts of significantamountofintracellularLDH,asindicatedbythe
facultatively anaerobic oral bacteria collected from saliva amountoftotalLDHrecoveredfromlysed,untreatedHGF
withexposuretoincreasingconcentrationsofKSL(Fig.4). (Fig.6b).
Fig.3.Killingof:(a)L.salivariusATCC29602;
(b) S.mutans ATCC 25175T; (c) S. gordonii
ATCC51656;and(d)Actinobacillusactinomy-
cetemcomitans ATCC 43718 by KSL. Killing
assaysweredoneinHBSSwiththeexceptionof
Actinobacillus actinomycetemcomitans, which
wasassayedin0.01%BHI.Pointsandvertical
linesrepresentthemeanandSDofquadruplicate
determinations.Thedatarepresenttheresultsof
one of the three separate experiments per-
formed.
1088 JournalofMedicalMicrobiology52
Bactericidaldecapeptide
Fig.4.Log reductions in c.f.u. of facultatively anaerobic bacteria
collectedfromsalivaafterincubationfor30minat378Cin10mM
PPBwithorwithoutKSL(control,dH O).AMann–Whitneytestwas
2
used for comparison of the experimental groups with the control
group. The asterisk represents statistical significance from control
(P,0.05).Thedatarepresenttheresultsofoneofthethreeseparate
experiments,eachperformedinquadruplicate.Bars,SD.
DISCUSSION
Inthisstudy,wetestedthegrowth-inhibitoryactivityofKSL,
a cationic decapeptide that was identified by synthetic
combinatorial library technology (Hong et al., 1998). We
have demonstrated that this peptide: (i) was effective in
inhibitingthegrowthofabroadrangeoflaboratorystrainsof
oral bacteria; (ii) at higher concentrations, was capable of
reducingviablecountsofmembersofresidentsalivabacteria
collectedfromhealthyhumansubjects;and(iii)didnotshow
anycytotoxiceffectsagainstHGF.
Formostoralbacteriatested,effectivekillingdosesofKSLin Fig.5.LIVE/DEADBacLightstainingofsalivabacteriatreatedwith
vitroarecomparabletothoseofotherantimicrobialpeptides, (a)dH O(control)or(b)KSL.Manyoftheisolatedbacteriaappearin
2
which include human defensins (Miyasaki et al., 1990; Raj aggregatesofvarioussizes.Note:manybacteriainthecontrolsample
etal.,2000b),rabbitbactenecins(Rajetal.,2000a),human stainedgreen(live),whereasasignificantnumberofbacteriainthe
salivary histatins (Helmerhorst et al., 1997, 1999; MacKay KSL-treatedsamplestainedred(dead).Thebrightredovalstructure,
et al., 1984) and porcine protegrins (Miyasaki et al., 1997, locatednearthetopontherightoftheimageforthecontrolsample,
1998;Moscaetal.,2000).OurstudiesshowedthatKSLwas representsthenucleusofadeadbuccalepithelialcell.Magnification,
effectiveagainstmanyoralpathogens.Forexample,growth 4003.
of Actinomyces naeslundii, a putative pathogen that is
involvedinthedevelopment ofgingivitis (Loesche&Syed,
1978)androot-surfacecaries(Summey&Jordan,1974)and
is an early colonizer for plaque formation (Kolenbrander bacterial species exhibit a wide range of susceptibilities to
etal.,1993;Gibbons,1996)wasinhibitedbyKSLatafairly antimicrobial peptides (Boman, 1991; Zasloff, 2002). The
lowconcentration,i.e.3.13(cid:1)gml(cid:1)1.Similarly,growthofS. basisforthedifferenceinsusceptibilitiesofbacterialspecies
mutansATCC25175T andL.acidophilus,cariogenicorgan- againstparticularpeptidesisnotknownatpresent(Zasloff,
isms(Bowden,1991;vanHoute,1994),wasinhibitedbyKSL 2002).
atconcentrationsof,10(cid:1)gml(cid:1)1.
Bycontrast,growthofothermembersoftheS.oralisgroup,
In this study, growth of S.oralis [amemberof theS.oralis S. sanguinis,S. gordoniiand S.mitis, was inhibited byKSL,
group (Kilian et al., 1989)] and Actinobacillus actinomyce- withMICsthatrangedfrom25to50(cid:1)gml(cid:1)1.Membersof
temcomitans appeared to be less affected by KSL in our the S. oralis group, together with Actinomyces naeslundiias
susceptibilityassay.Atpresent,wedonotknowwhatfactors describedabove,areprimarycolonizersofthecleanedtooth
maycontributetovariationofsusceptibilityamongdifferent andcanundergointra-andintergenericco-aggregationwith
strains of oral bacteria tested. However, it is known that a range of partner organisms in vivo, which contributes to
http://jmm.sgmjournals.org 1089
S.P.Concannonandothers
InadditiontothedeterminationofMICsandMBCsofKSL
againstoralbacteria,wealsoperformedabactericidalassay,
i.e.logreductionsinviablecounts,toobtainED valuesof
99
KSL on selected organisms. These oral bacteria, which
included L. salivarius, S. mutans, S. gordonii and Actinoba-
cillusactinomycetemcomitans,showedsignificantreductions
inviability(.3logs)whenexposedtoKSLatconcentrations
of ,10(cid:1)gml(cid:1)1. With the exception of Actinobacillus
actinomycetemcomitans[acausativeagentforjuvenileperio-
dontitis(Slotsetal.,1986)]andS.gordonii,ourobservations
generallysupportedtheMICsthatweredeterminedforthese
selected organisms. Interestingly, whilst KSL was less effec-
tive in inhibiting the growth of Actinobacillus actinomyce-
temcomitans and S. gordonii, as shown by the broth
microdilution assay for MIC determinations, the peptide
showedmorepotentinhibitoryactivityagainstthegrowthof
theseorganismsinthebactericidalassay.Thesediscrepancies
couldbeattributabletothesalinityofassaymediausedfor
determining the MICs and ED of KSL, as indicated by a
99
numberofearlierreportsoninvitrobactericidalactivityof
antimicrobialpeptides(Leeetal.,1997;Friedrichetal.,1999;
Tanakaetal.,2000;Guthmilleretal.,2001;Murakamietal.,
2002;Zasloff,2002).
In this study, we were also interested in establishing the
conformation of KSL in membrane environments. The
three-dimensional structure of this antimicrobial peptide
has not been reported previously, although the secondary
structureofthispeptidewaspredictedbycirculardichroism
Fig.6.Determinationofreductionof(a)MTTand(b)LDHreleaseby (CD). Previous CD data suggested an Æ-helical conforma-
KSL-treatedoruntreatedfibroblasts.Pointsandbarsrepresentthe
tionforKSLinthepresenceof50%trifluoroethanolanda
meanandSDoftriplicatedeterminations.Twothousandfivehundred distortedÆ-helicalstructureinthepresenceof25mMSDS
andtenthousandfibroblastswereusedineachdeterminationofLDH
(Ohetal.,1999).However,determinationofthehelicityof
releaseandMTTreduction,respectively.Cellswereculturedfor72h
smalloligopeptidesbasedonabsolutemeanellipticityvalues
at378CinaCO incubatorbeforeexposuretodifferentconcentra-
2
oftenleadstoambiguoussecondarystructureprediction(Raj
tionsofKSL.HGFwereexposedtoKSLfor6hpriortomeasure-
etal.,1990).Moreover,theconformationalfeaturesofeach
ments. The data represent the results of one of the two separate
experiments. individualresidueinthesequenceofthepeptidecannotbe
ascertainedandacleardistinctionbetweenÆ-and3 -helical
10
structures cannot be made by CD data. Hence, we deter-
mined the three-dimensional structure of KSL both in
aqueous solution and in DMSO, which mimics the polar
aproticmembraneenvironment.NMRdataindicatethatthis
plaqueaccumulation(Kolenbrander&London,1992).The peptideremainslargelyinitsÆ-helicalconformation(Fig.2)
observationthatKSLat50(cid:1)gml(cid:1)1 orbelowwasinhibitory
in membrane environments, whilst it prefers to adopt an
in vitro to growth of most members of the S. oralis group,
unfoldedrandomstructureinaqueoussolution.
Actinomycesnaeslundiiandthemajorcariogenicpathogens
suggests that KSL could be effective in controlling the TheviewofthehelicalstructureofKSLalongthehelixaxis
formation of plaque and dental caries in vivo. This is (Fig. 2) does not reflect a perfect amphiphilic structure.
supportedbytheobservationthatKSL,athigherconcentra- Enhancing amphiphilicity by the substitution of Lys1 and
tions, caused significant reductions in viable counts of Lys8 by Leu residues has been reported to increase the
residentsalivabacteria.Thisisparticularlyrelevantassaliva helicity. However, antimicrobial activity has been found to
bacteriaarereleasedfrombiofilmsformedonhardandsoft decrease(Ohetal.,1999),therebyindicatingtheimportance
tissuesintheoralcavity(Helmerhorstetal.,1999).Further- ofcationicresiduesandtheweakamphiphilicityofKSL.The
more, it has been shown that these bacteria or bacteria weak amphiphilic nature of KSL indicates that its sponta-
collected from plaque are more resistant to antimicrobial neousinsertionintomicrobialmembranesandformationof
peptides than pure cultures of oral bacteria (Helmerhorst ion channels across cell membranes is unlikely. This weak
et al., 1999). This may be one of the reasons why higher amphiphilicityprobablyaccountsfortheminimaltoxicityto
concentrations of antimicrobials are needed to inhibit the mammaliancellsobservedforKSL.Thispeptideispolarand
growthofresidentsalivabacteria,asalsoshowninthisstudy. hydrophilic, suggesting that the mechanism of its antimi-
1090 JournalofMedicalMicrobiology52
Bactericidaldecapeptide
crobial action could primarily involve electrostatic (ionic REFERENCES
type),hydrogenbondingandhydrophobicinteractionswith Bateman,A.,Singh,A.,Shustik,C.,Mars,W.M.&Solomon,S.(1991).
thepolarfaceofmicrobialmembranesorwithamembrane- The isolation and identification of multiple forms of the neutrophil
bound receptor molecule, leading to possible membrane granule peptides from human leukemic cells. J Biol Chem 266,
damage. 7524–7530.
Blondelle, S. E. & Houghten, R. A. (1996). Novel antimicrobial
Furthermore, in the initial characterization of this novel compounds identified using synthetic combinatorial library technol-
antimicrobialpeptide,Hongetal.(1998)suggestedthatthis ogy.TrendsBiotechnol14,60–65.
decapeptide may kill micro-organisms by attacking their Blondelle,S.E.,Takahashi,E.,Weber,P.A.&Houghten,R.A.(1994).
membranes,asextrapolatedfromtheirworkperformedwith Identificationofantimicrobialpeptidesbyusingcombinatoriallibraries
artificial membranes. By using a BacLight Viability kit, we madeupofunnaturalaminoacids.AntimicrobAgentsChemother38,
2280–2286.
demonstrated that KSL could cause actual membrane
damage to bacteria. This was illustrated by the presence of Blondelle,S.E.,Pe´rez-Paya´,E.&Houghten,R.A.(1996a).Synthetic
combinatorial libraries: novel discovery strategy for identification of
asignificantnumberoforganismsthatfluorescedredinthe
antimicrobialagents.AntimicrobAgentsChemother40,1067–1071.
KSL-treatedsalivabacteriasample.SYTO9,whichispresent
Blondelle, S. E., Takahashi, E., Houghten, R. A. & Pe´rez-Paya´, E.
intheassaysolution,canenterallcellsandfluorescesgreen.
(1996b). Rapid identification of compounds with enhanced antimi-
Ontheotherhand,propidiumiodide,whichfluorescesred
crobial activity by using conformationally defined combinatorial
and is present as the second stain in the assay solution, is
libraries.BiochemJ313,141–147.
excluded from cells with intact membranes. However,
Boggiano, C., Reixach, N., Pinilla, C. & Blondelle, S. E. (2003).
propidium iodide is able to enter cells with a damaged
Successfulidentificationofnovelagentstocontrolinfectiousdiseases
membrane and competes with and quenches SYTO 9 for fromscreeningmixture-basedpeptidecombinatoriallibrariesincom-
labelling the DNA, to make such cells fluoresce red (Lisle plexcell-basedbioassays.Biopolymers71,103–116.
et al., 1999). The staining reactions of the bacteria that we Boman,H.G.(1991).Antibacterialpeptides:keycomponentsneededin
observedprovidestrongmorphologicalevidencetosuggest immunity.Cell65,205–207.
that the membranes of KSL-treated bacteria were compro- Boman, H. G. (1998). Gene-encoded peptide antibiotics and the
mised, whereas membranes of bacteria exposed to dH O conceptofinnateimmunity:anupdatereview.ScandJImmunol48,
2
remainedintact,asillustratedbythepresenceofmanygreen 15–25.
fluorescentbacteriainthissample. Bowden,G.H.W.(1991).Whichbacteriaarecariogenicinhumans?In
RiskMarkersforOralDiseases,vol.1,pp.266–286.EditedbyN.W.
AnearlierstudyshowedthatKSLisnon-toxictomammalian Johnson.Cambridge,UK:CambridgeUniversityPress.
cells, as demonstrated by its lack of haemolytic activity Chen,J.,Falla,T.J.,Liu,H.&9otherauthors(2000).Developmentof
againstmouseerythrocytes(Hongetal.,1998).Inthisstudy, protegrinsforthetreatmentandpreventionoforalmucositis:structure-
we confirmed and extended the in vitro toxicity study to activityrelationshipsofsyntheticprotegrinanalogues.Biopolymers55,
88–98.
include testing of KSL against HGF. Our data indicated
strongly that KSL neither induced cell death nor compro- Davies,J.(1994).Inactivationofantibioticsandthedisseminationof
resistancegenes.Science264,375–382.
misedthemembraneintegrityofHGFthatwereexposedto
anuptotenfoldexcessofeffectivebactericidaldosagesofthis Decker, T. & Lohmann-Matthes, M. L. (1988). A quick and simple
methodforthequantitationoflactatedehydrogenasereleaseinmeas-
peptide.Similarlytootherantimicrobialpeptides(Hancock,
urements of cellular cytotoxicity and tumor necrosis factor (TNF)
1997b;Zasloff,2002),theresultssuggestthatKSLspecifically
activity.JImmunolMethods115,61–69.
targets the membrane of prokaryotes, but not that of
Dyson,H.J.&Wright,P.E.(1991).Definingsolutionconformationsof
mammaliancells.
smalllinearpeptides.AnnuRevBiophysBiophysChem20,519–538.
Elsbach,P.(2003).Whatistherealroleofantimicrobialpolypeptides
In this study, we have focused on the effects of KSL on
that canmediateseveralotherinflammatory responses?JClinInvest
cariogenic organisms and primary colonizers that are in-
111,1643–1645.
volved in the early development of dental plaque. Another
Friedrich,C.,Scott,M.G.,Karunaratne,N.,Yan,H.&Hancock,R.E.W.
important step towards the establishment of KSL as a
(1999). Salt-resistant alpha-helical cationic antimicrobial peptides.
potential antiplaque agent is to test, in future studies, the
AntimicrobAgentsChemother43,1542–1548.
effectiveness of KSL against subgingival plaque bacteria,
Fuchs,P.C.,Barry,A.L.&Brown,S.D.(1998).Invitroantimicrobial
which are primarily obligate anaerobes and consist of
activityofMSI-78,amagaininanalog.AntimicrobAgentsChemother42,
organisms that are involved in the development of perio- 1213–1216.
dontaldiseases(Socransky,1998).
Gibbons,R.J.(1996).Roleofadhesioninmicrobialcolonizationofhost
tissues:acontributionoforalmicrobiology.JDentRes75,866–870.
Guthmiller, J. M., Vargas, K. G., Srikantha, R., Schomberg, L. L.,
ACKNOWLEDGEMENTS
Weistroffer,P.L.,McCray,P.B.,Jr&Tack,B.F.(2001).Susceptibilities
of oral bacteria and yeast to mammalian cathelicidins. Antimicrob
This work was supported by the US Army Medical Research and
AgentsChemother45,3216–3219.
MaterielCommand.Theviewsexpressedinthisarticlearethoseofthe
Hancock, R. E. W. (1997a). Antibacterial peptides and the outer
authors and do not reflect the official policy or position of the
Department of the Army, the Department of Defense or the US membranesofGram-negativebacilli.JMedMicrobiol46,1–3.
Government. Hancock,R.E.W.(1997b).Peptideantibiotics.Lancet349,418–422.
http://jmm.sgmjournals.org 1091